Figure 3.

Decrease in B-Myb reduces clathrin on the mitotic spindles and leads to mitotic arrest. (A) Decrease in B-Myb reduces clathrin signals on the mitotic spindles. HeLa cells were treated with a control luciferase siRNA (upper panels) or B-myb siRNA (lower panels), subjected to indirect immunofluorescent staining with anti-clathrin HC (Cy3, red), and anti-α-tubulin (Alexa Fluor 488, green). DNA was stained with TOTO-3 iodide (blue) and cells were visualized using confocal microscopy. Scale bars: 10 μm. On the right, the clathrin signal at the mitotic spindle was quantified and indicated by bar graph with ±s.d. ^**P<0.01. A value of one represents no specific signal at the mitotic spindle (see Materials and methods). (B) B-Myb depletion decreased clathrin light chain (CLC) signal localization to the mitotic spindle. HeLa cells expressing EGFP-CLC were isolated, and were then transfected with B-myb siRNA or control luciferase siRNA. Cells were extracted with 1% Triton X-100 for 10 min prior to fixation, and immunostained with anti-GFP and visualized using confocal microscopy. Scale bars: 10 μm. (C) B-Myb depletion enhances mitotic arrest. The mitotic incidence of HeLa cells transfected with B-myb siRNA or control luciferase siRNA are indicated in the bar graph. (D, E) B-Myb depletion caused thicker metaphase plates and an increase in misaligned chromosomes. HeLa cells transfected with B-myb siRNA or control luciferase siRNA were fixed, washed with 0.2% Triton X-100 for 10 min, and immunostained with anti-Aurora B or anti-CENPB (Cy3, red), anti-α-tubulin (Alexa Fluor 488, green), and TOTO-3 iodide (blue), and visualized using confocal microscopy. A typical example is shown. The misaligned chromosomes (arrow) and thickness of the metaphase plates are indicated. Scale bars: 10 μm.